c-kit<low pluripotent hemopoietic stem cells form CFU-S on day 16

Using Ly5 congenic mice, we characterized the early differentiation step of pluripotent hemopoietic stem cells. Lineage (Lin(-))/CD71(-) cells in the bone marrow cells were separated into major histocompatibility complex (MHC) class I-high/c-kit(low) and MHC class I-high/c-kit(less-than-low) populations from C57BL/6 Ly5.1 male mice. These two populations (1,000 cells) were transplanted into lethally irradiated (5.5 Gy x 2) C57BL/6 Ly5.2 female mice. Colony-forming unit in spleen (CFU-S) assays were carried out on days 10, 12, 14, 16, and 20. In the mice that received c-kit(low) cells, CFU-S were first detected on day 12, and the CFU-S counts gradually increased. In contrast, no visible colony was detected until day 14 in the mice that received c-kit(less-than-low) cells; CFU-S were first observed on day 16. Donor-derived (Ly5.1(+)) cells, such as B cells, T cells, and myeloid cells, were detected by fluorescence-activated cell sorter analyses, and donor-derived erythroid cells were detected by polymerase chain reaction analyses using Y-chromosome-specific primers. Donor-derived cells in the recipients of c-kit(low) cells were detected in the spleen, bone marrow, and peripheral blood on day 12 after transplantation, while they were detected on day 16 in the mice that received c-kit(less-than-low) cells. Therefore, c-kit(less-than-low) cells have the capacity not only to form CFU-S on day 16 but also to reconstitute the recipients with donor-derived hematolymphoid cells 16 days after transplantation.